Mechanistic study on ultrasound assisted

Ultrasonics Sonochemistry 28 (2016) 207–217

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Mechanistic study on ultrasound assisted pretreatment of sugarcane bagasse using metal salt with hydrogen peroxide for bioethanol production Govindarajan Ramadoss, Karuppan Muthukumar ⇑ Department of Chemical Engineering, Alagappa College of Technology Campus, Anna University, Chennai 600 025, India

a r t i c l e

i n f o

Article history: Received 24 December 2014 Received in revised form 28 June 2015 Accepted 9 July 2015 Available online 10 July 2015 Keywords: Sugarcane bagasse Ultrasound Metal salt Holocellulose recovery Delignification Bioethanol

a b s t r a c t This study presents the ultrasound assisted pretreatment of sugarcane bagasse (SCB) using metal salt with hydrogen peroxide for bioethanol production. Among the different metal salts used, maximum holocellulose recovery and delignification were achieved with ultrasound assisted titanium dioxide (TiO2) pretreatment (UATP) system. At optimum conditions (1% H2O2, 4 g SCB dosage, 60 min sonication time, 2:100 M ratio of metal salt and H2O2, 75 °C, 50% ultrasound amplitude and 70% ultrasound duty cycle), 94.98 ± 1.11% holocellulose recovery and 78.72 ± 0.86% delignification were observed. The pretreated SCB was subjected to dilute acid hydrolysis using 0.25% H2SO4 and maximum xylose, glucose and arabinose concentration obtained were 10.94 ± 0.35 g/L, 14.86 ± 0.12 g/L and 2.52 ± 0.27 g/L, respectively. The inhibitors production was found to be very less (0.93 ± 0.11 g/L furfural and 0.76 ± 0.62 g/L acetic acid) and the maximum theoretical yield of glucose and hemicellulose conversion attained were 85.8% and 77%, respectively. The fermentation was carried out using Saccharomyces cerevisiae and at the end of 72 h, 0.468 g bioethanol/g holocellulose was achieved. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis of pretreated SCB was made and its morphology was studied using scanning electron microscopy (SEM). The compounds formed during the pretreatment were identified using gas chromatography–mass spectrometry (GC–MS) analysis. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Bioethanol can be produced from lignocellulosic biomass (LCB) such as crop residues (wheat straw, rice straw, corn stover, sugarcane bagasse (SCB), rice hulls, barley straw, sweet sorghum bagasse, olive stones), hardwood, softwood, cellulose wastes, herbaceous biomass and municipal solid wastes [1]. Among the various renewable energy sources, SCB is considered as an overabundant biomass because 5.4 108 dry tons of sugarcane is processed annually throughout the world. SCB is mainly composed of cellulose, hemicellulose and lignin. The holocellulose (cellulose + hemicellulose) recovery during the pretreatment is an important criterion, which enhances the fermentable sugar production during hydrolysis. In order to obtain high sugar yield, the protective coats around cellulose, hemicellulose and lignin need to be altered or detached without affecting the sugars during the pretreatment.

⇑ Corresponding author. E-mail address: [email protected] (K. Muthukumar). http://dx.doi.org/10.1016/j.ultsonch.2015.07.006 1350-4177/Ó 2015 Elsevier B.V. All rights reserved.

The pretreatment techniques studied include acid, alkaline, biological pretreatment, wet oxidation, organosolv, ozonolysis, ultrasound pretreatment and hydrogen peroxide with metal salts pretreatment. These methods are significantly different from one another in terms of reaction conditions, process efficiency and complexity. The ultrasonic pretreatment produces sonochemical and mechanoacoustic effects which affect the chemical and physical composition of SCB. The mechanoacoustic effect alters the surface structure of the biomass, whereas sonochemical effect produces hydroxyl radicals, which attack the components of SCB [2]. The pretreatment of SCB with metal salts and hydrogen peroxide (H2O2) was found to enhance the rate and yield of hydrolysis [3–5]. This method is considered as an attractive process, because the metal salts are recyclable and less corrosive than inorganic acids [4]. The decomposition of lignin occurs due to the formation of hydroxyl radicals and superoxide ions during this process [6]. The incorporation of ultrasound with this method is expected to alter the morphological structure of SCB, disrupt its structure to achieve effective hydrolytic process, and reduces the mass transfer limitations.

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G. Ramadoss, K. Muthukumar / Ultrasonics Sonochemistry 28 (2016) 207–217

In this study, SCB was pretreated with ultrasound in the presence of different metal salts (manganese sulphate monohydrate (MnSO4H2O), zinc oxide (ZnO), ferrous sulphate heptahydrate (FeSO47H2O), ammonium molybdate ((NH4)6Mo7O24), cobalt chloride (CoCl2), titanium dioxide (TiO2)). Based on higher holocellulose recovery and delignification, best metal salt was selected and the optimization of different process parameters such as H2O2 concentration, SCB dosage, ultrasound time, molar ratio of metal salt and H2O2, temperature, ultrasound amplitude and ultrasound duty cycle was carried out.

filtered using Whatman filter paper, washed several times with distilled water, hot water and finally with acetone to remove the residual metal salt present. The filtered SCB was dried to 60 °C until constant weight was observed, and then it was subjected to dilute acid hydrolysis. The experiments were carried out in duplicate and average values were reported. The presence of reducing sugars in the liquid phase was determined by DNS method. The % holocellulose recovery in the pretreated SCB was calculated using the following equation

% Holocellulose recovery ¼ 2. Materials and methods 2.1. Materials The experiments were conducted using SCB collected from local sugarcane processing unit located at Chennai, India. It was washed thoroughly with distilled water to remove dust and soluble sugars, dried at 60 °C for 24 h. The dried SCB was subsequently milled, screened to a particle size of below 1 mm (>18 mesh size, ASTM standard) and stored in an air tight polyethylene bag at room temperature. The chemicals such as acetone, ammonium molybdate, 3,5-dinitrosalicylic acid, cobalt chloride and titanium dioxide, were purchased from Sisco Research Laboratory (P) Ltd., Mumbai, India. Ferrous sulphate heptahydrate and manganese sulphate monohydrate were purchased from Central Drug House (P) Ltd., New Delhi, India. Sulphuric acid was purchased from Thermo Fischer Scientific India, Ltd., Mumbai, India, Hydrogen peroxide (30%) was purchased from Merck Specialities (P) Ltd., Mumbai, India and zinc oxide was purchased from Sigma–Aldrich chemicals (P) Ltd., Bangalore, India. All the chemicals used were of analytical grade. 2.2. Sonicator Sonotrode H3 probe type, made up of titanium with tip diameter 3 mm and length 100 mm (Hielscher Ultrasonic Processor UP400S, Germany) was used in this study. The operating power and frequency of the sonicator were 400 W and 24 kHz, respectively. The temperature was controlled using a water bath. 2.3. Pretreatment The schematic diagram of the experimental setup used in this study is shown in Fig. S1 (See Supplementary data). SCB was pretreated using ultrasound assisted metal salt pretreatment. Initially 2 g of SCB was placed in a 250 mL Erlenmeyer flask with desired concentration of H2O2 and metal salt. The contents were irradiated with ultrasound at different operating conditions. Metal salts such as manganese sulphate monohydrate (MnSO4H2O), zinc oxide (ZnO), ferrous sulphate heptahydrate (FeSO47H2O), ammonium molybdate ((NH4)6Mo7O24), cobalt chloride (CoCl2), and titanium dioxide (TiO2) were used. The effect of operating parameters such as H2O2 concentration (0.25%, 0.50%, 1% and 2% v/v), SCB dosage (1, 2, 3 and 4 g), sonication time (15, 30, 45 and 60 min), molar ratio of metal salts to H2O2 (0.5:100, 1:100, 2:100 and 4:100 g/mL), temperature (25, 50, 75 and 100 °C), ultrasound amplitude (30%, 40%, 50% and 60%) and ultrasound duty cycle (50%, 60%, 70% and 80%) were investigated for maximum holocellulose recovery and delignification. The performance of ultrasound assisted metal salt pretreatment was compared with ultrasound pretreatment carried out at 100 °C with 2 g SCB for 60 min. The optimization of operating parameters was done by changing one variable at a time and operating conditions were fixed based on the literature [7]. The pretreated SCB was

HRPT-SCB 100 HRSCB

ð1Þ

where HRSCB is the amount of holocellulose in native SCB and HRPT-SCB is the amount of holocellulose in pretreated SCB expressed in (g/g). The % delignification in the pretreated SCB was calculated using the following equation

% Delignification ¼

DSCB DPT-SCB 100 DSCB

ð2Þ

where DSCB is the amount of lignin present in native SCB and DPT-SCB is the amount of lignin present in pretreated SCB expressed in (g/g). 2.3.1. Calorimetric efficiency The actual power dissipated into the system (calorimetric efficiency) using ultrasound probe was determined by calorimetric method. The rise in temperature of a fixed quantity of water in an insulated container for a given time was measured. Then, the actual energy (power) dissipated into the liquid was calculated from the following equation:

W¼

mC p DT t

ð3Þ

where M is the mass of solution taken in ‘kg’, Cp is the specific heat of liquid at constant pressure (4.180 J/kg K), DT is the difference between initial and final temperature during the ultrasound time in ‘K’, and t is the reaction time in ‘s’ [8]. Acoustic intensity was calculated using the equation given below:

I¼

Actual power dissipated ðWÞ Area of horn tip ðm2 Þ

ð4Þ

For the ultrasound assisted titanium dioxide pretreatment system (UATP), the reaction mixture was subjected to ultrasound irradiation using an ultrasonic horn made up of titanium (tip diameter of 0.3 102 m) operated at 24 kHz with a rated output power of 400 W and the surface area of the ultrasound irradiating face was 7 106 m2. The horn was operated at 50% amplitude for 60 min with 7 s on and 3 s off duty cycle. The difference in temperature during the course of the reaction was observed as 24.6 K and the corresponding power dissipation and acoustic intensity were found to be 2.856 W and 408 kW/m2. 2.4. Hydroxyl radicals measurement The formation of free radicals during the pretreatment of SCB using ultrasound assisted metal salt played an important role in delignification. The radicals formed were quantified using Fricke dosimetry method [9]. Fricke solution was prepared using 1 mM FeSO4, 0.4 M H2SO4 and 1 mM NaCl and this was added into the reaction mixture containing SCB, H2O2 and metal salt. The reaction was carried out as per the pretreatment conditions. The samples were withdrawn at regular time intervals and the formation of Fe3+ during the pretreatment was determined using Elico double beam SL210 UV–Visible spectrophotometer at 304 nm. The estimation of hydroxyl radical is based on the oxidation of Fe2+ to Fe3+.


The number of hydroxyl radicals formed in Fricke solution is theoretically equal to one quarter the amount of Fe3+ produced [10].

2.5. Dilute acid hydrolysis About 1 g of pretreated SCB with maximum holocellulose content was placed in an Erlenmeyer flask (250 mL) and added with desired concentration of sulphuric acid. The sulphuric acid concentration was varied as 0.25%, 0.5%, 1% and 2% v/v and experiments were carried out at constant liquid to solid ratio (LSR) of 20:1 mL/g and at 121 °C for 15 min. The reaction mixture was cooled to room temperature after the hydrolysis and the samples were centrifuged at 8000 rpm for 10 min. Then the supernatant was subjected to glucose, xylose, arabinose, acetic acid and furfural analysis using HPLC [11]. The acid concentration was optimized based on the yield of sugar. The hydrolyzate obtained was added with sodium hydroxide (2 N) to make the solution neutral for ethanol fermentation. All experiments were carried out in triplicate and average values were reported. The catalytic efficiency [E] of dilute acid hydrolysis of pretreated SCB was calculated, using the following equation:

P S P ½E ¼ 1þ I

ð5Þ

P where S is the sum of the sugar concentrations in the hydrolyzate P (glucose, xylose and arabinose) and I is the sum of the inhibitor concentrations in the hydrolyzate (acetic acid and furfural) [12].

2.6. Fermentation The neutralized hydrolyzate was subjected to fermentation using Saccharomyces cerevisiae. About 100 mL of hydrolyzate was placed in a 250 mL Erlenmeyer flask and it was supplemented with CaCl2 (0.025 g/L), KH2PO4 (25 g/L) MgCl2 (0.025 g/L), peptone (5 g/L) and yeast extract (3 g/L). The contents were sterilized at 121 °C for 15 min and allowed to cool. Then, inoculated with 24 h old seed culture of S. cerevisiae maintained at 30 °C. The samples withdrawn at regular time intervals were subjected to ethanol analysis.

2.7. Analytical methods SCB constituents such as cellulose, hemicellulose, lignin and ash were determined by detergent extraction method as described in our previous study [13,14]. The reducing sugars content was determined by 3,5-dinitrosalicyclic acid (DNS) method [15]. Glucose, xylose, arabinose, acetic acid and ethanol were determined using high performance liquid chromatography (Agilent 1260 Infinity binary pump with RI detector and Hi-Plex H column (300 7.7 mm)) operated with 5 mM H2SO4 as mobile-phase at a flow rate of 0.6 mL/min [16]. Furfural was detected using UV chromatograms at 280 nm. High resolution GC–MS systems (JEOL GC MATE II, USA) with quadruple double focusing mass analyzer and capillary HP-5 ms column were used. High pure helium was used as carrier gas at a flow rate of 1 mL/min. The column oven temperature was programmed between 50 and 250 °C with a progressive rise of 10 °C/min. The ion source and gas interface temperature of mass spectrometer detector was held at 250 °C and the ions were separated based on their mass to charge (m/z) ratio in the range of 50– 500. The National Institute of Standards and Technology (NIST) mass spectral library and a custom library of known compounds were used for the identification of compounds.

209

2.7.1. Fourier–transform infrared spectroscopy To determine the changes in the functional groups of SCB before and after the pretreatment, Fourier-transform infrared spectroscopy (FTIR) (Perkin-Elmer FTIR Spectrophotometer 2000 series) was used with detector range at 4 cm1 resolution and 25 scan per sample. The pellets were prepared using 2 mg of dried, powdered and pretreated biomass and 200 mg of KBr. The spectra were recorded between 4000 and 550 cm1. 2.7.2. X-ray diffraction The crystalline and amorphous nature of SCB constituents before and after pretreatment was examined using X-ray diffractometer (XRD, PANalytical X’Pert PRO) with the applied potential and current of 40 kV and 30 mA, respectively. The samples were scanned in the 2h range of 5–35° with a step size of 0.05° and the crystallinity index (CrI) was obtained from the relationship between the intensity of cellulose (I002) peak and the minimum dip (Iam) peaks using the equation given below:

CrIð%Þ ¼

I002 Iam 100 I002

ð6Þ

where I002 is the maximum intensity of the 0 0 2 lattice diffraction and Iam = intensity of the amorphous portion at 18.8°, 2h degrees [17]. 2.7.3. Scanning electron microscopy The structural modification of native, pretreated and dilute acid hydrolyzed SCB was observed using VEGA 3 TESCAN model (Czech Republic). Samples were adhered to double sided carbon tape on brass sample stubs and sputter coated with gold (sputter EMITECH SC 7620 model). This assembly was kept in a vacuum desiccator till the analysis was made. Sample images were observed with an accelerating voltage of 10 kV and SCB samples were examined at different magnifications ranging from 800 to 1400. 3. Results and discussion 3.1. Effect of ultrasound with metal salt pretreatment SCB used in this investigation was found to contain 38.0 ± 0.4% cellulose, 32.0 ± 0.2% hemicellulose, 27.0 ± 0.1% lignin, 2.3 ± 0.3% ash and 0.7 ± 0.3% others. The influence of different metal salts on ultrasound assisted pretreatment of SCB was studied and the results are shown in Fig. 1. The ultrasound pretreatment gave 70.12% cellulose recovery, 60.3% hemicellulose recovery and 35.7% delignification at 100 °C, 50% ultrasound amplitude, 70% ultrasound duty cycle and 60 min total course of time. The holocellulose recovery observed at these conditions was 65.6%. It is clear that the application of ultrasound alone showed low cellulose recovery and delignification. The ultrasound assisted H2O2 pretreatment showed 78% cellulose recovery, 29% hemicellulose recovery, 38.17% delignification and holocellulose recovery of 55.6%. Among the different metal salts, TiO2 gave maximum holocellulose recovery of 80.94 ± 1.00% and delignification of 66.67 ± 1.79%. This may be due to the synergistic effect of ultrasound and TiO2 which caused the disintegration of carbohydrate–lignin linkage to release lignin and hemicellulose [18]. TiO2 acts as a sonocatalyst because the TiO2 particles intensify the hydroxyl radicals generation [19]. Ultrasound irradiation of water is known to cause acoustic cavitations, which induce millions of small bubbles to collapse and to achieve temperature more than 5000 K and pressures as high as 100 MPa. As a result, generation of high-speed microjets (physical effect) and hydroxyl radicals occurs due to the pyrolysis

210


% Holocellulose recovery/ Delignification

90

Holocellulose recovery Delignification

80 70

60 50

2.6-fold more production than ultrasound assisted ammonium molybdate pretreatment, which gave least performance on delignification among the metal salt used for pretreatment. In the case of ultrasound pretreatment with H2O2, OH formation was found to be 0.0438 mM/min, which was higher than FeSO47H2O and (NH4)6Mo7O24 pretreatment with ultrasound as shown in Fig. 2b and this was further confirmed based on the corresponding delignification value. 3.2. Effect of hydrogen peroxide

40 30 20 10 0 A

B

C

D

E

F

G

Effect of different pretreatment Fig. 1. Effect of different pretreatment methods on holocellulose recovery. A – ultrasound + H2O2 [conditions (1% H2O2, 2 g SCB dosage, 60 min ultrasound time, 100 °C, 50% ultrasound amplitude and 70% ultrasound duty cycle)], [B – ultrasound + H2O2 + TiO2, C – ultrasound + H2O2 + MnSO, D – ultrasound + H2O2 + ZnO, E – ultrasound + H2O2 + CoCl2, F – ultrasound + H2O2 + FeSO4, G – ultrasound + H2O2 + (NH4)6Mo7O24. [conditions (1% H2O2, 2 g SCB dosage, 60 min ultrasound time, 100 °C, 1:100 (molar ratio of metal salt and H2O2), 50% ultrasound amplitude and 70% ultrasound duty cycle]].

of water (chemical effect) [20]. In addition, the TiO2 particles act as additional nucleus which further increases the cavitation under ultrasonic irradiation [21]. Moreover, thermal excitation of TiO2 by extremely high temperatures resulting from the cavitation bubble implosions, lead to the generation of more hydroxyl radicals. The aromatic ring structure of lignin opens at a-position due to the cleavage of C–C linkage [22]. During the pretreatment, the C–H linkage is susceptible to break and form macroradicals, which in turn react with H and OH produced by cavitation. This might be responsible for the induction of depolymerization process [23]. Ultrasound assisted TiO2 pretreatment (UATP) solubilized 18.78% hemicellulose and 19.34% cellulose and the pretreatment liquid showed 12.28% reducing sugar. The order for the maximum holocellulose recovery and delignification with respect to other salts was MnSO4H2O > ZnO > CoCl2 > FeSO47H2O > (NH4)6Mo7O24. The lower holocellulose recovery and delignification value may be due to lesser hydroxyl radical formation and this was confirmed by Fricke dosimetry. The results showed almost similar delignification for MnSO4H2O and ZnO and the corresponding values were 50.14 ± 1.26% and 48.88 ± 1.49%, respectively. Manganese salts reported to enhance delignification due to its high catalytic activity [6]. Similarly the combined effect of MnSO4H2O and ZnO salts with H2O2 gave higher delignification [14]. But in the case of FeSO47H2O and (NH4)6Mo7O24, the delignification was lesser than the value of ultrasound pretreated SCB and it might be due to the disruption of lignin and hemicelluloses. The dissolved lignin and carbohydrate oligomers forms condensation and precipitation onto the pretreated biomass [24,25]. Hydroxyl radical is a powerful oxidant and plays a vital role in oxidative delignification. It initiates the degradation of lignin through diffusion limited rate reactions [26]. Fig. 2a presents the effect of sonication time on Fe3+ production and it shows a linear increase with an increase in time. The maximum production of Fe3+ ions indicates maximum production of OH. In this study, the maximum OH formation of 0.0736 mM/min was observed with UATP process (1% H2O2, 2 g SCB, 60 min sonication time, 100 °C and 1:100 M ratio of metal salt and H2O2). This showed

Fig. 3A presents the effect of H2O2 concentration on holocellulose recovery and delignification during UATP. In this study, H2O2 was used for the generation of hydroxyl radicals. The oxidation potential of hydroxyl radicals (2.8 V) is much greater than other oxidizing agents such as ozone (2.07 V) and chlorine (1.39 V). Ultrasound catalyzed the dissociation of hydrogen peroxide into hydroxyl radicals through chain reactions. These oxidants react with complex propyl phenyl chains of lignin and convert them into simple phenols. It should be noted that H2O2 itself act as a hydroxyl radical ‘scavengers’, which reduce the delignification efficiency during oxidation of lignin. Hence, the optimization of hydrogen peroxide dosage is important. In this study, concentration of H2O2 was varied and the delignification values obtained were 30.25 ± 0.64 (0.25% H2O2), 45.33 ± 2.10 (0.5% H2O2), 66.67 ± 1.19 (1% H2O2) and 68.41 ± 1.34 (2% H2O2). The results showed two fold increase with 1% H2O2 compared to 0.25% H2O2. But in the case of 2% H2O2, marginal increase in delignification was observed. This may be due to the scavenging activity of H2O2 present in the reaction mixture. The results showed 80.94 ± 1.00% and 78.84 ± 0.14% holocellulose recovery for 1% and 2% H2O2, respectively. The data indicated that increase in H2O2 concentration up to 1%, increased the holocellulose recovery and further increase did not influence the holocellulose recovery. The decrease in holocellulose recovery at 2% H2O2 may be due to the formation of reducing sugars from hemicellulose as metal salts have the tendency to hydrolyze pentose sugar more easily than hexose sugar [4]. The reducing sugar concentration in the liquid obtained after the pretreatment was found to be 7.6% and 8.4% for 1% and 2% H2O2, respectively. From these results, 1% H2O2 was considered as the optimum concentration and similar results were observed during the treatment of newspaper with H2O2 [7]. 3.3. Effect of substrate dosage The effect of substrate dosage was studied by varying the SCB dosage from 1 to 4 g. The results obtained are shown in Fig. 3B. The holocellulose recovery obtained for 1, 2, 3 and 4 g of SCB were 72.03 ± 1.71%, 80.94 ± 1.00%, 84.45 ± 0.81% and 88.37 ± 1.03%, respectively and the corresponding delignification values were 70.18 ± 0.23%, 66.67 ± 1.19%, 63.62 ± 1.43% and 62.77 ± 0.66%, respectively. It was inferred from the results that the increase in SCB dosage increased the holocellulose recovery and decreased the delignification. The synergistic effect of TiO2 and H2O2 in the presence of ultrasound favored maximum holocellulose recovery at 4 g of substrate dosage. The maximum delignification was observed at low substrate dosage, and this may be due to the more interaction of TiO2 and H2O2 with SCB. This leads to the formation of hydronium ion (H3O+) and OH that enhance the rate of depolymerization of lignin [27]. At lower substrate dosage (1 g), the presence of radicals tends to solubilize the hemicellulosic and part of cellulosic fractions, which gave highest reducing sugar yield of 13.8%. The least yield (5.1%) was observed for 4 g. In general, the cavitational effect increases due to the presence of solids, but higher concentration of solids increases the viscosity and affects


211

Fig. 2. (a) Effect of reaction time on Fe3+ concentration for ultrasound assisted metal salt pretreatment system. (b) Effect of different pretreatment process on hydroxyl radical production rate for ultrasound assisted metal salt pretreatment system. A – ultrasound + H2O2 + (NH4)6Mo7O24, B – ultrasound + H2O2 + FeSO4, C – ultrasound + H2O2, D – ultrasound + H2O2 + CoCl2, E – ultrasound + H2O2 + ZnO, F – ultrasound + H2O2 + MnSO4, G – ultrasound + H2O2 + TiO2 [conditions for A, B, D, E, F and G (1% H2O2, 2 g SCB dosage, 60 min ultrasound time, 100 °C, 1:100 (molar ratio of metal salt and H2O2), 50% ultrasound amplitude and 70% ultrasound duty cycle] [conditions for C (1% H2O2, 2 g SCB dosage, 60 min ultrasound time, 100 °C, 50% ultrasound amplitude and 70% ultrasound duty cycle].

mass and heat transfer characteristics, which in turn affected the delignification at higher substrate dosages (4 g). 3.4. Effect of sonication time In order to find the optimum sonication time, the experiments were carried out at different sonication time ranging from 15 to 60 min. Fig. 3C shows the effect of sonication time on holocellulose recovery and delignification. The holocellulose recovery found at the end of 15, 30, 45 and 60 min were 78.16 ± 0.14%, 82.47 ± 1.07%, 85.62 ± 0.22% and 88.37 ± 0.97%, respectively and delignification observed were 52.98 ± 1.27%, 55.87 ± 2.11%, 60.50 ± 0.70% and 62.77 ± 0.66%, respectively. The increase in sonication time increased the holocellulose recovery and delignification and this may be due to the disruption of ether bonds between lignin and hemicellulose [28,29]. The cavitational effects increased with an increase in time and such conditions results in improvement of mass transfer and reaction rates [30]. The experiments were carried out for 75 min and cellulose recovery and delignification obtained at the end of 75 min was 88.99 ± 0.23% and 61.2 ± 0.17%, respectively. However, there was not much significant difference in cellulose recovery and delignification value at 75 min compared to 60 min. Hence, the sonication time of 60 min was considered as the optimum time for the UATP system. Similar results were observed during the treatment of wheat straw using ultrasound [29]. The reducing sugar concentration obtained at the end of 15, 30, 45 and 60 min were found to be 9.8%, 8.2%, 6.9% and 5.1%, respectively. 3.5. Effect of molar ratio of metal salt and H2O2 The effect of molar ratio of metal salt to H2O2 was studied and the results obtained are shown in Fig. 3D. The pretreatment was carried out at optimum H2O2 concentration, substrate dosage and sonication time of 1%, 4 g SCB and 60 min, respectively. The molar ratio of TiO2 to H2O2 was varied as 0.5:100, 1:100, 2:100 and 4:100. The maximum holocellulose recovery and delignification of 92.75 ± 0.74% and 72.06 ± 1.34%, respectively, were observed for 2:100 M ratio. For lower molar ratio (0.5:100), holocellulose recovery and delignification values obtained were 80.41 ± 2.11% and 58.64 ± 0.78%, respectively. The increase in molar ratio increased the holocellulose recovery and delignification up to 2:100 M ratio and further increase did not show significant effect. The reducing sugar yield of 8.9%, 5.1%, 3.6% and 12.12% were obtained for 0.5:100, 1:100, 2:100 and 4:100 M ratio, respectively. At high molar ratio (4:100), the metal salt present in the reaction mixture

dissociated and produced more cations, which enhanced the fast hydrolysis of hemicellulose and hence, the pretreated liquid contains more reducing sugars [31]. The holocellulose recovery is an important criterion for fermentable sugar production and maximum value was obtained with 2:100 M ratio. Hence further studies were carried out with 2:100.

3.6. Effect of temperature To study the effect of temperature, experiments were carried out at different temperatures ranging from 25 to 100 °C and remaining parameters were maintained at their optimum values (1% H2O2, 4 g SCB, 60 min and 2:100 M ratio of metal salt and H2O2). The results obtained are shown in Fig. 3E. The holocellulose recovery observed at 25, 50, 75 and 100 °C were 80.26 ± 1.78%, 86.37 ± 0.69%, 94.98 ± 1.11% and 92.75 ± 0.74%, respectively and the corresponding delignification values were 60.83 ± 0.24%, 71.45 ± 1.02%, 78.72 ± 0.86% and 72.06 ± 1.34%, respectively. There was an increasing trend in both holocellulose recovery and delignification with respect to temperature. This may be due to the formation of more amount of free radical at higher temperature, which initiated the breakdown of covalent bonds leading to higher reaction rates [32]. The increase in operating temperature beyond 75 °C decreased the holocellulose recovery and delignification. The increase in temperature affect the collision of cavitational bubbles produced during the compressive phase of sonication. Bubble collision is well connected with the production of shock waves, which are responsible for the myriad effect of ultrasound. High temperature helps to disrupt the strong solute matrix which encompasses van der Waals forces, hydrogen bonding and dipole attraction. Moreover diffusion rates are high at higher temperatures. Nevertheless, cavitation effects are better achieved at low temperatures when the ultrasonic power is constant. This may be due to the fact that, as the temperature increases, the vapor pressure of the solvent also increases, which facilitates more solvent vapor to fill the cavitation bubbles and this in turn collapse less violently. Hence the sonication effects are less intense at higher temperatures [33]. In this study, 75 °C was found to be the most favorable temperature for both cellulose recovery and delignification, because further increase in temperature leads to the production of inhibitors and degradation of carbohydrates, which eventually decreased the sugar yield. This observation is in agreement with the results obtained for the pretreatment of sustainable raw material using ultrasound assisted alkaline pretreatment [34].

212


Fig. 3. Effect of pretreatment parameters on holocellulose recovery and delignification for ultrasound assisted TiO2 system.

213


3.7. Effect of ultrasound amplitude The influence of ultrasonic amplitude on the holocellulose recovery and delignification was investigated. The experiments were carried out at different amplitudes ranging from 30% to 60% and the results obtained are shown in Fig. 3F. The holocellulose recovery obtained for 30%, 40%, 50% and 60% amplitude were found to be 81.24 ± 0.62%, 88 ± 0.98%, 94.98 ± 0.47% and 95 ± 0.52%, respectively, and the corresponding delignification values were 61.43 ± 1.09%, 69.14 ± 1.02%, 78.72 ± 0.86% and 76.28 ± 0.47%, respectively. The increase in ultrasound amplitude from 30% to 50% increased the holocellulose recovery and delignification. The further increase in amplitude to 60% did not show significant effect. The higher amplitude decreased the delignification and this may be due to the adverse cavitational effects [35]. The formation of huge number of cavitational bubbles at the tip of the ultrasound transducer at higher amplitude may obstruct the transfer of energy from the transducer to the liquid medium. Hence, 50% was arrived as the optimum.

Table 1 Comparison of different pretreatment methods reported in the literature. Biomass

Pretreatment

SCB

Ultrasound assisted ammonia pretreatment 10% ammonia at 80 °C for 45 min and LSR 1:10 (g/mL) Dual salt pretreatment with H2O2 1% H2O2, 1 g SCB, 0.5:100 g/mL molar ratio of metal salt (MnSO4H2O and ZnO) and H2O2 at 100 °C for 30 min Non-ionic surfactant-dilute ammonia pretreatment SCB, ammonium hydroxide (28% v/v solution), and water at a ratio of 1:0.5:20; 3% (w/w) surfactant at 160 °C for 1 h Tween 80 PEG 4000 Ionic liquid pretreatment 0.6 g NH4OH–H2O2 pretreated SCB was incubated in 19.4 g of [Amim]Cl at 100 °C for 6 h Ultrasound assisted alkali pretreatment 2% NaOH solution at 50 °C for 20 min and LSR of 20:1 (mL/g) Alkaline hydroxide pretreatment 6% H2O2 at 20 °C for 4 h

SCB

SCB

SCB

3.8. Effect of ultrasound duty cycle Ultrasound duty cycle is defined as the fraction of time for which the ultrasonic system is operated. For example, duty cycle of 50% indicates the application of ultrasound for 5 s, which was followed by a rest period (no ultrasound) for 5 s. The effect of different ultrasound duty cycle (50%, 60%, 70% and 80%) on holocellulose recovery and delignification was investigated and the results obtained are shown in Fig. 3G. The maximum holocellulose recovery and delignification of 94.98 ± 0.47% and 78.72 ± 0.86%, respectively, were observed with 70% ultrasound duty cycle. The holocellulose recovery and delignification increased with an increase in duty cycle from 50% to 70% and further increase to 80% did not show any effect. The increase in delignification might be due to the increase in time of exposure to ultrasonic irradiation, which in turn increase the cavitational bubbles formed and also increase the implosion [36]. The employment of continuous mode ultrasound leads to corrosion and unnecessary increase in electrical energy consumption compared to pulsed mode of operation. In this study, the maximum holocellulose recovery and delignification was observed with 70% duty cycle. The results obtained were in agreement with the results reported for the pretreatment of sustainable raw material using ultrasound assisted alkaline pretreatment [34]. 3.9. Comparison of different pretreatment techniques Table 1 presents the cellulose recovery and delignification of SCB obtained from various pretreatment methods [11,14,37–42]. Compared to the reported pretreatment methods, UATP system showed better results for cellulose recovery and delignification. This may be due to the synergistic effect of ultrasound and TiO2 in the presence of H2O2 which degraded the complex phenolic groups of lignin. 3.10. Dilute acid hydrolysis The SCB pretreated at optimized pretreatment conditions (1% H2O2, 4 g SCB dosage, 60 min ultrasound time, 2:100 M ratio of metal salt and H2O2, 75 °C, 50% ultrasound amplitude and 70% ultrasound duty cycle) was chosen for dilute sulphuric acid hydrolysis. In order to obtain maximum monosaccharide sugars from pentose and hexose sugars, pretreated SCB was hydrolyzed with dilute H2SO4 ranging from 0.25% to 2%. Table 2 presents the soluble fraction of SCB constituents obtained from dilute H2SO4 hydrolysis. The major component obtained from cellulose and hemicellulose

SCB

SCB

SCB

SCB

SCB

Dilute mixed acid pretreatment 1% (H2SO4 and CH3COOH) at 190 °C and LSR 1:10 g/mL for 10 min Ultrasound assisted alkaline pretreatment 2% NaOH solution at 40 °C for 1 h and LSR of 20:1 (mL/g) Ultrasound assisted titanium dioxide pretreatment (UATP) system 4 g SCB, 1% H2O2, 60 min at 75 °C and 2:100 M ratio of metal salt and H2O2

Cellulose recovery and delignification

Refs.

95.78% and 58.14%

[11]

93.42% and 74.18%

[14]

[37]

87% and 37% 86% and 47% 86.1% and 30.69%

[38]

99% and 75.44%

[39]

88% (delignification alone)

[40]

85.4% and 4.7%

[41]

90.6% (delignification alone)

[42]

94.02% and 78.72%

Present work

was glucose and xylose, which constitutes about 80–90% of total fermentable sugar. The dilute acid hydrolysis was carried out at 121 °C for 15 min as described in our previous study [14]. The maximum concentration of xylose (10.94 ± 0.35 g/L) and arabinose (2.52 ± 0.27 g/L) were obtained with 0.25% H2SO4. At lower acid concentration, maximum hemicellulose conversion of about 87.5% was obtained with less inhibitor production. The increase in acid concentration to 2% H2SO4 reduced the hemicellulose conversion to 60.4%. It should be noted that inhibitors concentration reached maximum (furfural 3.13 ± 0.03 g/L and acetic acid 2.56 ± 0.15 g/L) at high acid concentration (2% H2SO4). A decrease in xylose concentration with an increase in acid concentration might be due to the decomposition of xylose to furfural and similar results were reported by Rodriguez- Chong et al., [12]. The acetic acid formation in the liquid hydrolyzate during dilute acid hydrolysis was mainly due to cleavage of ester and acetyl linkages present in SCB. Acetic acid inhibits the microbial growth when its concentration is in the range of 4–10 g/L, as it diffuses through the cell membrane and decreases intracellular pH, causing adverse effects to the metabolism of the microorganisms [12]. Furfural is also a growth inhibitor and in this study, hydrolysis at 0.25% H2SO4 gave 0.76 ± 0.62 g/L of acetic acid and 0.93 ± 0.11 g/L of furfural. From the results, it is evident that the inhibitor production

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Table 2 The effect of acid concentration on hydrolyzate composition. Hydrolysis condition

H2SO4 (%, v/v)

Glucose (g/L)

Xylose (g/L)

Arabinose (g/L)

Total sugars (g/L)

Acetic acid (g/L)

Furfural (g/L)

Total inhibitor production (g/L)

121 °C for 15 min

0.25 0.5 1 2

14.86 ± 0.12 15.87 ± 0.05 17.35 ± 0.34 16.86 ± 0.23

10.94 ± 0.35 9.90 ± 0.22 8.64 ± 0.40 7.78 ± 0.36

2.52 ± 0.27 2.22 ± 0.19 1.96 ± 0.11 1.52 ± 0.16

28.32 ± 0.74 27.99 ± 0.46 27.95 ± 0.85 26.16 ± 0.75

0.76 ± 0.62 1.36 ± 0.07 1.95 ± 0.23 2.56 ± 0.15

0.93 ± 0.11 1.96 ± 0.37 2.48 ± 0.31 3.13 ± 0.03

1.69 ± 0.73 3.32 ± 0.44 4.43 ± 0.54 5.69 ± 0.18

Table 3 Comparison of sugar and inhibitors production during hydrolysis of SCB and their catalytic efficiency. Feedstock

SCB SCB SCB SCB SCB SCB

Hydrolysis condition

2% sulphuric acid, 50 °C, 45 min, 1:20 g/mL 2% sulphuric acid, 15 min, 121 °C, LSR 1:20 g/mL 2% sulphuric acid, 122 °C for 22 min 1.25% v/v sulphuric acid, 121 °C for 2 min 0.045 M sulphuric acid, 170 °C for 10 min 0.25% sulphuric acid, 15 min, 121 °C, LSR 1:20 g/mL

Total sugars

Inhibitors

Glucose + xylose + arabinose (g/L)

Furfural (g/L)

Acetic acid (g/L)

HMF (g/L)

24.16 30.46 24.6 26.30 25.09 28.31

0.59 1.19 0.5 1.4 1.43 0.93

1 0.68 3.65 4.0 2.72 0.76

ND ND ND ND 0.11 ND

was very low in this study and hence, this pretreatment process is an effective one. The glucose concentration of 17.35 ± 0.34 g/L was obtained with 1% H2SO4 whereas on the other hand, 1.95 ± 0.23 g/L of acetic acid and 2.48 ± 0.31 g/L of furfural were observed with 1% H2SO4. The concentration of glucose increased with an increase in H2SO4 concentration up to 1% and declined with further increase in acid concentration. This may be due to the release of some toxic compounds or inhibitors. Similar results were observed by Gupta et al., [43]. In this study, maximum glucose production of 17.35 g/L was obtained with 1% H2SO4 which constitutes about 85.8% theoretical yield of glucose whereas maximum pentosan conversion of 77.03% was obtained with 0.25% H2SO4. From Table 2, it was inferred that maximum fermentable sugar (28.32 ± 0.74 g/L) and minimum inhibitor production (1.69 ± 0.73 g/L) were observed with 0.25% H2SO4 and hence this concentration was considered as the optimum condition for dilute acid hydrolysis. Similarly, minimum fermentable sugar production of 26.16 ± 0.75 g/L and maximum inhibitor production of 5.69 ± 0.18 g/L were obtained with 2% H2SO4. 3.11. Catalytic efficiency The catalytic efficiency [E] was calculated to compare the efficiency of SCB hydrolysis and the values obtained are shown in Table 3. The [E] value greater than 1.0 indicates better hydrolysis and formation of high concentration of sugars and low concentration of growth inhibitors [12,14]. The maximum [E] value of 10.52 was obtained for 0.25% H2SO4, whereas 2% H2SO4 showed minimum [E] value of 3.91. 3.12. GC–MS analysis Fig. S2 shows the total ion chromatogram (TIC) of degraded lignin compounds present in the liquid fraction of UATP system. The peak present in the chromatogram was identified by reference compounds and mass spectrum. From the chromatogram, guaiacyl glycerol-b-guaiacylether, coniferyl alcohol, vanillin and sinapyl alcohol were found to be the main degradation products of lignin.

Catalytic efficiency (E)

Refs.

9.32 10.6 4.78 4.11 4.77 10.52

[11] [14] [44] [45] [46] Present study

Similar results were reported by Owen et al., [47]. The identified compounds and their mass number were depicted in Table S1 (See Supplementary information). The synergistic effect of ultrasound with TiO2 promoted the lignin depolymerization into aromatic compounds. This is because of the free radicals generation from the cleavage of various a or b-O-4 linkages of aryl ether bonds between the phenyl propane units. Radicals such as hydroxyl, methoxy, carbonyl, carboxyl and methyl were among the possible reactive species favored depolymerization process. It is evident from the results that simple phenols such as phenol, methyl and dimethyl phenol as well as monomeric phenols such as guaiacol, catechol, vanillin, syringol, coniferyl and sinapyl alcohol were produced due to the action of demethylation (DME), demethoxylation (DMO), decarboxylation (DCB), dehydroxylation (DOH) and decarbonylation (DCO) reactions during the pretreatment of SCB [48]. It should be noted that some depolymerization compounds of lignin were not produced during the pretreatment, which may be due to the absence of specific reactive free radicals. The present work attempts to increase the delignification using hybrid pretreatment utilizing ultrasound, metal salt (TiO2) and H2O2. This method showed 78.72% of delignification whereas ultrasound pretreatment showed only 38.6% [11]. The result showed twofold increase in delignification, when hybrid method was used. TiO2 in the presence of H2O2 fragment the lignin structures into quinones and other organic chemicals [48]. Mante et al. investigated highly selective defunctionalization of monomeric phenols into simple phenols using TiO2 by pyrolysis. The formation of monomeric phenolics from lignin pyrolysis is accompanied by free radicals, which are generated due to the cleavage of different linkages and process condition. The free radicals and the aromatic ring undergo several secondary reactions to form various compounds such as methoxylated phenolics, hydroxyl phenols, methanol and water. Catechol and 4-methylcatechol were found to be reactive phenolics present in the product stream obtained from lignin pyrolysis using TiO2. The incorporation of ultrasound with TiO2 and H2O2, highly favored the formation of reactive phenolics such as guaiacol, phenol, and syringol, at 75 °C. On the other hand, the pyrolysis temperature was 550 °C, which is comparatively very high compared to ultrasound assisted TiO2 pretreatment (UATP).


Fig. 4. FTIR spectra of SCB pretreated at different optimized conditions for ultrasound assisted TiO2 pretreatment system. A – native SCB, B – (1% H2O2, 2 g SCB dosage, 60 min ultrasound time, 1:100, 100 °C, 50% ultrasound amplitude and 70% ultrasound duty cycle), C – (1% H2O2, 4 g SCB dosage, 60 min ultrasound time, 1:100, 100 °C, 50% ultrasound amplitude and 70% ultrasound duty cycle), D – (1% H2O2, 4 g SCB dosage, 60 min ultrasound time, 2:100, 100 °C, 50% ultrasound amplitude and 70% ultrasound duty cycle), E–G – (1% H2O2, 4 g SCB dosage, 60 min ultrasound time, 2:100, 75 °C, 50% ultrasound amplitude and 70% ultrasound duty cycle).

3.13. Characterization of native and pretreated SCB The FTIR spectra of native and pretreated SCB are shown in Fig. 4. The prominent band at 3334 cm1 was ascribed to the O– H stretching vibration and hydrogen bonding in phenolic and aliphatic structures. A sharp band present at 2901 cm1represents the C–H stretching of methyl and methylene groups. These regions can easily be differentiated in native and pretreated SCB. The absorption peak at 1729 cm1 was due to the stretching vibration of carbonyl groups (C@O) in uronic acids or acetyl groups attached to hemicelluloses [49]. The behavior of these spectra in the 1516– 1598 cm1 region shows all lignin sources, pertaining to the C–H bond deformations and aromatic ring vibration [50]. The weaker band at 1378 cm1 was attributed to aliphatic C–H stretching in methyl group. The enhancement of absorption spectra at 1240 cm1 belongs to C@O stretching vibration in lignin, xylan and ester groups. The peak at 1163 cm1 due to C–O–C stretching present at the b-(1–4) glycosidic linkage of cellobiose. The strong band at 1033 cm1 was assigned to C–O stretching of cellulose, hemicellulose and lignin or C–O–C stretching in cellulose and hemicelluloses. The increase in the peak intensity at 1033 cm1 represents the pretreated SCB showed higher holocellulose recovery from the native SCB. The signal at 899 cm1 was originated from the b-glucosidic linkages between sugar units in cellulose and hemicelluloses. The X-ray diffraction patterns of native and pretreated SCB are shown in Fig. S3 (See Supplementary data). The samples exhibited cellulose and hemicellulose diffraction peaks. The crystallinity index (CrI) of native SCB showed 49%, which increased to 70% after the pretreatment. There was an increase in the crystallinity index when SCB was subjected to pretreatment, which is due to the removal of hemicellulose and lignin. It was confirmed that the amorphous region of SCB was highly susceptible during the pretreatment and dilute acid hydrolysis. The major diffraction peak of cellulose was normally observed at 21.9°. Among the pretreatment methods, UATP showed better holocellulose recovery. The order of increase in the crystallinity index of pretreated SCB was

215

found to be E > D > C > B > A. It was clear that progressive increase in crystallinity index showed better results when SCB was pretreated with ultrasound assisted TiO2. Identical observations were reported earlier for the pretreatment of sugarcane bagasse using imidazolium ionic liquids [51]. The structural changes of SCB before and after the pretreatment were clearly observed using SEM. From Fig. 5, it can be noticed that the native SCB fibers showed compact rigid structure with smooth and continuous surface and the pretreated SCB depicted damaged internal surface. The UATP method partially removed lignocellulosic fraction such as hemicellulose and lignin. The smooth and continuous surface of native SCB showed lot of perforations on the surface after the pretreatment, which may be due to the synergistic action of ultrasound with metal salt. Similar results were reported in the literature for SCB and industrial hemps [52,53]. The perforations inflicted by the UATP process, may increase the accessible surface area of SCB to the acid catalyst for the betterment of acid hydrolysis. In this study, 85% of cellulose was converted to glucose during dilute acid hydrolysis within 15 min which showed the effectiveness of UATP process. 3.14. Mechanism of action Lignin is a complex heteropolymer and its molecular weight is greater than 500. During UATP, the formation of hydroxyl, methoxy and carbonyl radicals were enhanced due to the presence of TiO2, H2O2 and ultrasound. The lignin fragments were found to be converted into simple and monomeric phenols as shown in Fig. S4 (see Supplementary data). One of the lignin fragment (Molecular weight (MW ) =258) is converted into sinapyl alcohol by dehydroxylation (DOH). The formation of coniferyl alcohol from sinapyl alcohol may probably by demethoxylation (DMO) process. This study suggested that demethylation (DME) and dehydroxylation of sinapyl alcohol may be the direct pathway for the production of syringol (MW = 154). However, the formation of phenol (MW = 94) from guaiacol (MW = 124) and guaiacol from syringol (MW = 154) was highly favored by DMO. Another lignin fragment (MW = 211), a precursor for the production of vanillin, is produced by DMO and decarbonylation (DCO) process. The condensation of guaiacol and vanillin in the presence of hydroxyl and methyl radicals might be the strong reason for the generation of guaiacylgly cerol-b-guaiacyl ether (MW = 319). The freely available methyl radicals pairs with the phenolic group could lead to the formation of methyl and dimethyl phenol. The hydroxyl and methoxy moieties of phenolic group may adsorb on the TiO2 surface, which leads to the dissociation of bonds such as C–OH (DOH), C–OCH3 (DMO) and C–CH3 (DME), respectively [48]. 3.15. Fermentation The SCB hydrolyzate containing 14.86 ± 0.12 g/L of glucose, 10.94 ± 0.35 g/L of xylose and 2.52 ± 0.27 g/L of arabinose was fermented using S. cerevisiae. The maximum ethanol production observed at the end of 72 h was 13.24 g/L and is equivalent to 91.68% of theoretical yield. The ethanol production was found to be 0.468 g/g sugar and the result showed that UATP as a suitable method for ethanol production from SCB. 3.16. Commercial viability of the developed process The developed countries such as the United States and European countries such as Norway, Germany, Russia and Spain have developed pilot scale production and further commercialized the production of ethanol. According to US Department of Energy, cellulosic ethanol mitigates greenhouse gas emissions (GHG) by 85% over reformulated gasoline. By contrast, starch ethanol (from

216


Fig. 5. Scanning electron microscopy images of SCB (A) native SCB, (B) ultrasound assisted TiO2 pretreatment of SCB, conditions [1% H2O2, 4 g SCB, 60 min, 2:100 (molar ratio of metal salt and H2O2), 75 °C, 50% ultrasound amplitude and 70% ultrasound cycle].

corn), which usually uses natural gas to provide energy for the process, reduces greenhouse gas emissions by 18–29% over gasoline. It is interesting to note that ethanol produced from agricultural residues or cellulosic crops has significantly lower greenhouse gas emissions and a higher sustainability rating than ethanol produced from grain. In steam explosion pretreatment, part of the hemicellulose present in the biomass gets hydrolyzed and form acids, which catalyzes the hydrolysis of hemicellulose. High energy requirement and inhibitor generation are the major drawbacks of this process [54]. In the case of ammonia fiber explosion, employment of high-pressure liquid ammonia operated at moderate temperatures (50–90 °C), followed by instantaneous pressure release leads to lignin depolymerization, hemicellulose solubilization, and cellulose decrystallization. The cost of liquid ammonia, process handling, and recovery are the main bottlenecks [55]. The energy required for recompression of ammonia is also significantly high. Alkaline pretreatment using sodium hydroxide is effective in removing lignin and acetyl groups. However, it is not suitable for woody biomass due to its higher lignin content. Alkaline pretreatment improves the subsequent enzymatic hydrolysis of the pre-treated biomass, but it is a slow process. This requires neutralization, and the added alkali also needs to be recovered [56]. The pretreated biomass then hydrolyzed using acid or enzyme catalyst for the production of fermentable sugars. Dilute acid hydrolysis (0.7–3.0%) requires high operating temperatures (200–240 °C) and produces a large number of inhibitors like furans, organic acids, and phenolics. The usage of concentrated acid requires high amounts of acid and hence becomes uneconomical. The acid recycling also entails considerable costs [56]. The foregoing analysis indicated that the UATP system produced only less inhibitors with higher holocellulose recovery and delignification. Usage of chemicals also very limited and requires less time. These factors make this process as an advantageous compared to the above mentioned pretreatment process in terms of process efficiency, inhibitor generation and pretreatment time. 4. Conclusion This study presented ultrasound assisted metal salt pretreatment of SCB for improved bioethanol production. The UATP of SCB gave maximum holocellulose recovery and delignification. The operating parameters were optimized and at optimum

conditions, 94.98 ± 1.11% of holocellulose recovery and 78.72 ± 0.86% of delignification were observed. Among the different salts studied in the presence of ultrasound, ammonium molybdate showed least performance and TiO2 showed better performance. The major products observed with UATP system include vanillin, syringol, coniferyl alcohol, sinapyl alcohol, guaiacol, etc., The maximum catalytic efficiency and sugar yield of 10.52 and 28.32 g/L were observed with 0.25% H2SO4 at the operating conditions of 15 min, 121 °C and LSR of 20:1 mL g1. At the end of 72 h fermentation, the hydrolyzate produced 13.24 g/L of bioethanol which is equivalent to 91.68% of theoretical yield. Hence, it can be concluded that UATP system as a suitable method for the pretreatment of SCB to produce bioethanol. Acknowledgments The authors gratefully acknowledge Council of Scientific and Industrial Research (CSIR), New Delhi, India for providing financial support to carry out this research work under CSIR Project scheme (Grant No. 22(0564)/12/EMR-II). One of the authors, Mr. G. Ramadoss, is grateful to CSIR, New Delhi for the award of CSIR-SRF. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ultsonch.2015.07. 006. References [1] C.E. Wyman, Handbook on Bioethanol: Production and Utilization, Taylor & Francis, Washington D.C., 1996. [2] M.J. Bussemaker, D. Zhang, Effect of ultrasound on lignocellulosic biomass as a pretreatment for biorefinery and biofuel applications, Ind. Eng. Chem. Res. 52 (2013) 3563–3580. [3] C. Liu, C.E. Wyman, The enhancement of xylose monomer and xylotriose degradation by inorganic salts in aqueous solutions at 180 °C, Carbohydr. Res. 341 (2006) 2550–2556. [4] L. Liu, J. Sun, C. Cai, S. Wang, H. Pei, J. Zhang, Corn stover pretreatment by inorganic salts and its effect on hemicellulose and cellulose degradation, Bioresour. Technol. 100 (2009) 5865–5871. [5] Q. Yu, X. Zhuang, Z. Yuan, W. Qi, Q. Wang, X. Tan, The effect of metal salts on the decomposition of sweet sorghum bagasse in flow-through liquid hot water, Bioresour. Technol. 102 (2011) 3445–3450. [6] R. Agnemo, G. Gellerstedt, The reactions of lignin with alkaline hydrogen peroxide. Part II. Factors influencing the decomposition of phenolic structures, Acta Chem. Scand. B 33 (1979) 337–342.

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